The rat as an experimental animal.

The development and characterization of many inbred, congenic, and recombinant strains of rats in recent years has led to the detailed genetic description of this species, especially in regard to its major histocompatibility complex. This information has contributed substantially to the study of comparative genetics and has greatly enhanced the utility of the rat in a variety of areas of biomedical research. This article focuses on the use of the rat in immunogenetics, transplantation, cancer-risk assessment, cardiovascular diseases, and behavior.

Effect of hyperlipemic serum on cholesterol accumulation in monkey aortic medial cells.

The effect of hyperlipemic monkey serum on cholesterol ester formation and accumulation in monkey aortic medial cells grown in tissue culture was studied. The cellular incorporation and esterification of free cholesterol was followed using the specific activity of serum labeled with free [14C]cholesterol while the cellular sterol content was analyzed by gas-liquid chromatographic techniqyes. The effects produced by hyperlipemic monkey serum (HMS) and normal monkey serum (NMS) were evaluated at both comparable percentage levels in the media and at equivalent exogenous cholesterol concentrations. When the two sera were adjusted to equal exogenous free cholesterol levels, the incorporation of free cholesterol by the aortic medial cells was related to the free cholesterol concentration of the culture media whether supplied by normal or hyperlipemic serum cholesterol. Under these conditions the total cholesterol content of the HMS-grown cells was 35% greater than that of NMS-grown cells, due to an elevation in free cholesterol of approximately 3 mug/mg cell protein and a 2- to 4-fold increase in esterified cholesterol. At similar percentage levels, the hyperlipemic serum stimulated a greater incorporation of free cholesterol into the monkey medial cells, accompanied by a 2-fold increase in the cellular esterification of this free cholesterol.

Growth of rabbit aortic smooth-muscle cells cultured in media containing diabetic and hyperlipemic serum.

Smooth-muscle cell cultures were grown from thoracic aortas of normal and diabetic rabbits. The effect of diabetic rabbit serum on the growth of these cultures was studied both in the first, rapid-growth phase and the following, more "stationary" phase of growth. Control experiments were carried out on normal sera to which glucose had been added. The concentrations of cholesterol, phospholipid, and triglyceride were same in both normal and diabetic sera. Media containing diabetic serum stimulated the growth of cultures significantly in both phases (2p less than 0.01). This occurred in experiments utilizing cells from normal as well as from diabetic rabbits. Control media containing normal serum with added glucose had no such effect. The growth-promoting effects of diabetic serum and of hyperlipemic serum from nondiabetic rabbits were of the same order of magnitude. Autoradiographic studies showed that the number of 3H-thymidine-labeled cells increased significantly after culture in diabetic serum (2p less than 0.005). Cells cultured from the very beginning in diabetic serum or normal serum with added glucose were significantly larger than cells grown in control serum (2p less than 0.05 and 2p less than 0.01, respectively). Cells grown in hyperlipemic serum were significantly smaller than those grown in normal serum (2p less than 0.01). These results indicate that diabetic serum contains a factor or factors that stimulate the arterial medial cell to excessive growth. This factor is not glucose, insulin, or lipid. The results may be of relevance for the understanding of human diabetic macroangiopathy.

Reticuloendothelial system response to hyperlipidemia in rhesus and cynomolgus monkeys.

A detailed study of the effect of various periods of hyperlipidemia on the reticuloendothelial system (RES) lipid accumulation in rhesus and cynomolgus monkeys was conducted. The cynomolgus serum cholesterol and triglyceride levels were on the average more elevated than the rhesus levels throughout a 12-month period when both species were fed a diet containing 12.5% coconut oil, 12.5% butter fat, and 2% cholesterol. After cynomolgus monkeys were fed this diet, their reticuloendothelial system became more lipid laden than that of the rhesus monkeys, in both the liver and the spleen. This was also true for the circulating monocytes. Furthermore, the parenchymal cells of the cynomolgus livers also become more fat filled, and chemical analyses demonstrated more cholesterol (total, free, and esterified) and triglycerides in the liver and the spleen. Xanthomata development in the cynomolgus, although similar in type and distribution, was more extensive than that in the rhesus monkey after similar periods of experimental diet feeding. Therefore, the RES of two species of macaque monkeys are affected differently when challenged with the same high fat, high cholesterol diet, with the cynomolgus RES being much more involved with lipid and cholesterol storage than the rhesus RES.

New insights into the pathogenesis of atherosclerosis as revealed by PDAY. Pathobiological Determinants of Atherosclerosis in Youth.

In this progress report the major pathological results gained from the research program called The Pathological Determinants of Atherosclerosis in Youth (PDAY) are summarized. These results are made possible because of the many unique features of this multicenter study, which are also summarized. The following main accomplishments utilize special quantitative techniques to study cellular, chemical and molecular (genetic) features of the developing plaques in young people. These include for the first time: The greater incidence of early progressive lesions in selective apo E phenotypes. The greater incidence of progressive lesions in black youth with an apo B deletion genotype. The much higher concentration of epitopes of oxidized LDL in smokers than non-smokers. More prevalent macrophages and lymphocytes in the standardized thoracic aortic samples, where lesions progress slowly, than in the abdominal aortic core samples, where lesions are much more likely to become severe. A strong correlation between the mast cell population and the concentration of biogenic amines in the lesions. The location of Lp(a) specific antigens in these developing lesions as compared to apo B. The accumulation of extracellular lipid where progression of lesions is most rapid, with special emphasis on the effects of smoking. The correlation of modulation of the intimal smooth muscle cells with the sites where progression of the plaque is most frequent. Preliminary ultrastructural evidence of intimal platelet and leukocyte adherence and entrance into the intima of the thoracic aorta, where there is likely to be lack of progression of lesions. A review of the recently published biochemical evidence of the correlation of increased lesion cholesterol and collagen content in the abdominal aorta. The continuing studies and their implications are also summarized.

Apoprotein B quantification in rhesus and cynomolgus monkey atherosclerotic lesions.

The concept that much of the cholesterol deposition in atherosclerotic plaque development is provided by ingress of blood-derived apo B-rich lipoproteins into the arterial intima is given support by the study of arterial apo B accumulation. To compare the arterial wall level of immunoreactive apo B during the progression of diet-induced atherosclerosis in two widely used animal models of atherosclerosis, rhesus and cynomolgus monkeys were fed an atherogenic diet for 4, 8, and 12 months and their abdominal aortas quantitated for apo B. Apo B was extracted from aortic intima-media homogenates in two forms: Tris-buffer extractable or 'loosely bounds' and detergent (Triton X-100) extractable or 'tightly bound'. The aortic extracts were quantitated for apo B by radial immunodiffusion, using goat anti-rhesus apo B along with serum LDL standards of the appropriate species diluted in the two extract solutions. The control monkeys' aortas contained only buffer-extractable apo B. The atherosclerotic aortas of both species of monkeys progressively increased their levels of loosely bound and tightly bound apo B through 4, 8, and 12 months of atherogenic diet feeding, with the 8- and 12-month cynomolgus aortas containing much larger amounts of apo B than the rhesus aortas. These differences in aortic apo B content could be accounted for by the greater rate at which the cynomolgus atherosclerotic lesions developed at the later time points. When the total lesion apo B levels were correlated with representative morphometrically-quantitated histopathologic sections of the homogenized aortas, a highly significant correlation was seen between the total aortic apo B values and both the absolute area of the intimal lesions and the total area of oil red O stainable lipid in the lesions (P less than 0.001). These data indicate that as atherosclerotic lesions become larger and richer in lipid with progression of the disease, the amount of apo B-associated lipoproteins which are deposited unmetabolized in the lesions increases. These lipoproteins are increased in both the tightly bound and loosely bound forms.

Stimulation of proliferation in stationary primary cultures of monkey aortic smooth muscle cells. Part 2. Effect of varying concentrations of hyperlipemic serum and low density lipoproteins of varying dietary fat origins.

The outgrowth of medial explants of thoracic aorta from Rhesus monkeys has used to study the influence of hyperlipemic serum on cell proliferation. After 5-6 weeks of rapid growth in BME plus 10% normal serum, the cultures reach a stationary phase during which they show little mitotic activity. When it replaces 5% of the normal serum in the media, hyperlipemic serum induces another proliferative phase in the cultures, as measured by [3H1thymidine incorporation and increase in culture area. Low density lipoprotein (LDL) has the greatest stimulatory effect, while high density lipoprotein (HDL) has no effect. Hyperlipemic serum or its LDL still stimulates the cells even when diluted to achieve cholesterol levels comparable to the values with normal serum or LDL. Normal LDL has no effect, even when concentrated to increase its cholesterol level in the media. Thus it appears that hyperlipemic LDL has a stimulatory effect on arterial smooth muscle cells which does not depend on its higher lipid or cholesterol level.

Correlations of aortic histology with gross aortic atherosclerosis and metabolic measurements in diabetic and nondiabetic Macaca nigra.

We studied the aortic histology of 28 Macaca nigra males and females, from 6 to more than 20 years old, normal and manifesting various degrees of spontaneous diabetes. Correlations of several metabolic and hormonal indicators of diabetes severity with gross and microscopic findings in the aortas demonstrated direct associations with the severity of atherosclerosis. Mild to relatively severe aortic lesions were present. These monkeys showed many changes similar to those observed in medium and large arteries of diabetic humans. Intimal proliferation, prominent extracellular fibers as part of the intimal thickening, and lipid deposition--mostly in extracellular locations--were particularly evident. Significant relationships were observed when glucose clearance, insulin secretion, and fasting glucose levels were correlated with all aortic microscopic findings. Cholesterol concentrations had no correlation with the histological state of the aortas, and triglyceride levels correlated only with aortic lipid content and intimal thickness. Aortic pathologic changes increased with age; diabetics had significantly greater changes than nondiabetics. Macaca nigra can be useful in the study of how diabetes affects the development of atherosclerosis without the influence of an atherogenic diet.

Atherosclerosis in a rhesus monkey with genetic hypercholesterolemia and elevated plasma Lp(a).

A female rhesus monkey with a marked elevation of total plasma cholesterol LDL and Lp(a) while on a low-fat, low-cholesterol diet, died at 22 years of age. Her spontaneous hypercholesterolemia was related to a genetically determined LDL receptor deficiency (Scanu, A.M. et al., J. Lipid Res., 29 (1988) 1671). Autopsy revealed grossly visible multifocal to diffuse raised yellow plaques predominantly in the aorta and, to a lesser extent, in the coronary arteries. Microscopically, the plaques in the aorta and in the coronary arteries showed heavy lipid deposition. Some had features seen in advanced human atherosclerotic plaques, including a fibrous cap and a necrotic core. Immunohistochemical staining showed a co-localization of apo(a) with apo B in lesion sites, a pattern seen frequently in advanced human atherosclerotic plaques. Evidence of fibrinogen/fibrin in the plaque areas was also seen, but was not co-localised with either Lp(a) or apo B. This monkey developed progressive atherosclerosis which was not induced by diet, but rather was dependent on the LDL receptor deficiency with a possible contribution by the elevated plasma levels of Lp(a).

Evidence for more extensive deposits of epitopes of oxidized low density lipoprotein in aortas of young people with elevated serum thiocyanate levels. PDAY Research Group.

Elevated levels of low density lipoprotein (LDL) and smoking have long been recognized as risk factors for atherosclerosis and coronary heart disease (CHD). However, the mechanisms by which these factors contribute to the disease have not been fully elucidated. It has been postulated from in vitro studies using serum and LDL from smokers that smoking increases the oxidation of LDL, which in turn contributes to atherogenesis. We know of no direct evidence linking oxidized LDL (oxLDL) in human arteries to increased atherosclerosis in individuals who show elevated serum thiocyanate levels (HST) as an indicator of recent smoking. We have studied arterial samples from smokers micromorphometrically and found that 'smokers' have a greater area in which oxLDL can be identified in the early stages of the disease than do "nonsmokers', i.e., individuals with low serum thiocyanate levels (LST). This study demonstrates a positive correlation between the extent of oxLDL in the fatty streaks as well as the fatty plaques of standardized core sample areas of the thoracic and abdominal aortas of a sample group of young people, aged 15-34, who have evidence of recent smoking based on their postmortem serum thiocyanate levels.

A comparison of the quantitation of macrophage foam cell populations and the extent of apolipoprotein E deposition in developing atherosclerotic lesions in young people: high and low serum thiocyanate groups as an indication of smoking. PDAY Research Group. Pathobiological Determinants of Atherosclerosis in Youth.

Smoking is considered a major risk factor for the development and progression of atherosclerosis. The effects of apolipoprotein E (apo E) and macrophages in the pathogenesis and progression of human atherosclerosis have not been adequately elucidated even though they are frequent components of the diseased arterial intima. Anatomically standardized samples of arteries from young people whose risk factor indices indicated them as "smokers" or "non-smokers" have been studied micromorphometrically. It was found that smokers have a greater area in which apo E is deposited in the early stages of the disease than do non-smokers. Smokers also demonstrated greater "macrophage foam cell populations" than did non-smokers. The study also demonstrates a positive correlation between the number of macrophage foam cells and the extent of apo E deposition in the developing lesions of the thoracic and abdominal aortas of white men aged 30-34 years who have evidence of recent cigarette smoking as determined by their postmortem blood thiocyanate levels.

The proliferative effect of platelets and hyperlipidemic serum on stationary primary cultures.

Outgrowths from explants of aortic media which have become stationary in the presence of a medium containing 10% normal serum have been studied. Homologous hyperlipidemic serum and especially its low density lipoprotein fraction has been shown to induce a second episode of proliferation in these cultures. Cell proliferation was evaluated by direct measurement of cell colony size and/or incorporation of [3H]thymidine visualized by autoradiography. The possibility has been investigated that the increase in arterial smooth muscle cell proliferation produced by hyperlipidemic serum might actually be due to a platelet factor present in that serum. Platelet-poor and platelet-rich sera were prepared from hyperlipidemic donors and added in a concentration of 5% to the culture medium. Both were equally effective in inducing proliferation; on the other hand, the addition of platelets from either hyperlipidemic or normolipidemic animals had no additive effect. The proliferation-stimulating effect of hyperlipidemic serum occurred even when the stationary cultures were maintained in a medium containing platelet-poor plasma serum for 2 weeks, prior to the addition of hyperlipidemic serum also derived from platelet-poor plasma. It is concluded that the proliferative effect of hyperlipidemic serum on stationary primary cultures does not depend on the presence of platelet-derived material. The implications of these observations on plaque formation are discussed.

Experimental atherosclerosis in rabbits fed cholesterol-free diets. Part 12. Comparison of peanut and olive oils.

The atherogenicity of peanut oil is well established as is the fact that the structure of the component triglycerides of peanut oil influences its atherogenicity. This study was carried out to determine if the relative saturation of peanut oil was partly responsible for the observed effects. Rabbits were fed a semipurified, cholesterol-free diet containing 14% of North American peanut oil (iodine value, 100), South American peanut oil (iodine value, 110) or olive oil (iodine value, 83) for 8 months. Rabbits fed olive oil exhibited higher levels of serum and liver lipids than did the two peanut oil-fed groups but significantly lower levels of aortic atherosclerosis. The findings confirm earlier observations that the structure of a fat can have an affect on its atherosclerogenic potential that is independent of its level of unsaturation.

The effect of various dietary regimens and cholestyramine on aortic glycosaminoglycans during regression of atherosclerotic lesions in rhesus monkeys.

The effect of various diets on the aortic glycosaminoglycan (GAG) composition was studied in rhesus monkeys. Aortas were obtained from monkeys fed diets containing cholesterol and comparative fats including coconut oil--butter and peanut oil and with and without cholestyramine. Additional groups in each experiment were placed on regression diets of low-fat, low-cholesterol with and without cholestyramine. Further, an atherogenic diet of coconut oil--butter was alternated every 2 months with a diet enriched with corn oil. GAG isolated from intima and media--adventitia indicated slight variations in the concentration of total GAG among different dietary groups but major differences in the concentration of individual GAG. The concentrations of hyaluronic acid and heparan sulfate were generally greater in aortas of monkeys fed corn oil diets than in those fed coconut oil--butter or peanut oil diets. The concentration of dermatan suulfate generally decreased during regression of lesions induced by the saturated fat--cholesterol diet. Furthermore, the aortas of monkeys with lesions from feeding peanut oil showed higher levels of dermatan sulfate and lower levels of chondroitin 4-sulfate than the saturated fat-fed groups. The addition of cholestyramine enhanced the effects of regression. These observations show that the composition of GAG of the arterial wall can be influenced by various dietary programs and that GAG play a role in induction and regression of experimental atherosclerotic lesions.

Serum low density lipoproteins with mitogenic effect on cultured aortic smooth muscle cells.

Low density lipoprotein (LDL) subspecies of different size and lipid mass were isolated by density gradient ultracentrifugation from the serum of male rhesus monkeys (Macaca mulatta) fed both a low fat, low cholesterol commercial primate ration, and cholesterol-supplemented high-fat diets, as well as from the serum of human donors. The mitogenic effect of these lipoproteins was examined using primary cultures of rhesus aortic smooth muscle cells. It was observed that the smaller LDL (molecular weight 2.7 X 10(6) from normolipidemic monkeys and a small LDL (molecular weight 2.6 X 10(6) occurring in some normal human subjects exhibited no mitogenic action. In turn, the larger LDL subspecies (molecular weight greater than 3.0 X 10(6), and buoyant density less than 1.030 g/ml), whether from normolipidemic or hyperlipidemic monkeys, or from some normal human subjects, had a marked proliferative action. The results indicate that both hyperlipidemic and normal sera (both human and rhesus) contain mitogenic LDL species although in different amounts. LDL-III, the rhesus equivalent of human Lp(a) was not mitogenic despite its similarity on size and lipid composition to the stimulating particles. However, on the removal of most of its large sialic acid moiety, a clear mitogenic action was observed. The mechanisms responsible for the proliferative effect are unclear and may involve LDL mass, lipid composition, and surface charge although other speculations cannot at present be ruled out. Furthermore, since the small LDL subspecies of either rhesus or human origin were nonmitogenic and similar in mass to the LDL found in calf serum, the mitogenic response of the smooth muscle cells to large LDLs may depend on their early conditioning with the LDL of calf serum.

Cholesterol vehicle in experimental atherosclerosis. Part 18. Comparison of North American, African and South American peanut oils.

Peanut oils were obtained from the United States (NAPNO), Africa (APNO), and South America (SAPNO) and their effects on atherosclerosis in rabbits fed 2% cholesterol were determined. The major differences among the oils were in the content of oleic (NAPNO, 48.9%; APNO, 58.6%; SAPNO, 36.4%) and linoleic (NAPNO, 29.6%; APNO, 21.7%; SAPNO, 41.1%) acids. In a series of 4 experiments it was found that South American peanut oil was 7% more atherogenic than African peanut oil and 18% more atherogenic than North American peanut oil. American peanut oil was 14% more atherogenic that corn oil (CO). South American peanut oil gave highest serum and liver lipid levels. The differences in atherogenicity may be due to the structure of the triglycerides of the various peanut oils.

Regression of arteriosclerotic lesions in rhesus monkey aortas after regression diet. Scanning and transmission electron microscope observations of the endothelium.

Scanning and transmission microscopy were used to study aortic intima atherosclerotic lesions in Rhesus monkeys during both progression and regression phases. Scanning micrographs of severely atherosclerotic lesions showed areas of disjunctive endothelium and discontinuous basement membranes, frequently accompanied by red blood cells and other circulating elements adhering to the surface. Transmission micrographs also showed occasional areas of endothelial cell damage and loss with lipid-laden smooth muscle cells and foam cells beneath. Regressed lesions (affected by low-fat, low-cholesterol diet with or without cholestyramine) showed endothelial changes suggestive of reparative processes. Scanning micrographs showed flattened residual lesions with continous endothelial lining, while transmission microscopy disclosed interdigitated intercellular tight junctions and frequently reduplicated basement membranes.

Effects of feeding fish oil on the properties of lipoproteins isolated from rhesus monkeys consuming an atherogenic diet.

This study examined plasma lipids and lipoproteins of rhesus monkeys fed fish oil incorporated into a highly atherogenic diet containing saturated fat and cholesterol. The animals were fed diets containing 2% cholesterol and either 25% coconut oil (group I), 25% fish oil/coconut oil (1:1; group II), or 25% fish oil/coconut oil (3:1; group III) for 12 months (n = 8/group). Adding menhaden fish oil to the diet increased plasma eicosapentaenoic acid and docosahexaenoic acid and decreased plasma linoleic acid in animals fed the fish oil containing diets. Plasma concentrations of all lipoprotein fractions were decreased in the fish oil groups. VLDL isolated from group I animals exhibited beta-mobility on agarose gels but the VLDL from groups II and III animals did not. The group I VLDL was more highly enriched in cholesteryl ester than was VLDL from groups II and III. Group I LDL had a small but significant increase in cholesteryl ester content compared to group III LDL. No differences in HDL composition were observed in the 3 groups. At least 6 times less apo E was recovered in VLDL, IDL, and LDL from group III animals than from group I animals. Assuming 1 molecule of apo B per lipoprotein particle, there were 50% fewer VLDL, IDL, and LDL particles in group III than in group I animals. Group III also had significantly lower molar ratios of apo E/apo B in VLDL, IDL, and LDL than did group I animals. When VLDL from all 3 groups were incubated with J774 macrophages at equal protein concentrations, only the VLDL from the group I animals stimulated cholesterol esterification. Thus, introducing fish oil into an atherogenic diet reduced the number of VLDL, IDL and LDL particles in plasma by as much as 50%, reduced the cholesteryl ester content of the circulating lipoprotein, and reduced the ability of the VLDL to stimulate cholesterol esterification in macrophages.

Carbodimide coupling to formalin-fixed red blood cells. A direct passive hemagglutination test for anti-horse ferritin.

The water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, has been used to couple horse ferritin covalently to formalin-fixed, human red blood cells. This method has been optimized with respect to sensitivity, range, and reproducibility. The sensitized cells are stable for at least 6 months at 4 degrees C, and they may be stored frozen without additives or special precautions. Titers of 1/216 are routinely obtained with antisera to ferritin.

Update on the pathogenesis of atherosclerosis.

The major components of atherosclerotic plaque, ultimately responsible for clinical effects, are deposited lipids--mostly cholesteryl esters and cholesterol, derived largely from the lower-density lipoproteins of the blood--and proliferated, modified arterial smooth muscle cells with their synthesized connective tissue products. Advanced plaques vary widely in the proportion of the two components, but evidence indicates that lipid deposition--especially of lipoprotein elements--often occurs in the lesion-prone intimal areas of the artery prior to the buildup of smooth muscle cells. The 1980s were remarkably productive for investigators who study the pathogenesis of atherosclerosis. We now know of the many forms of lower-density lipoproteins, i.e., low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL), some of which are more likely to be associated with accelerated atherosclerosis and some of which are more likely to be influenced by diet. Among these forms of LDL and VLDL are LDL-1, beta-VLDL, and Lp(a). Work has been reported implicating various alterations of endothelial function in the permeability of the arterial endothelial barrier in the transport of these low-density, cholesterol-rich macromolecules. Of possibly greater interest is the developing evidence that such proliferation-stimulating molecules as platelet-derived growth factor (PDGF) can be produced by a number of cells likely to be involved in the progression of atherosclerotic plaque. In addition to platelets, these include activated monocytes and monocyte-derived macrophages, injured endothelial cells, and smooth muscle cells, which can undergo an autocrine conversion to PDGF synthesis--possibly stimulated by LDL from hyperlipidemic serum. Leukotrienes and other endothelium-associated regulatory molecules may also take part in the paracrine and autocrine mechanisms of stimulating smooth-muscle-cell proliferation. Additional recent developments that have led to a better understanding of atherosclerotic pathogenesis have occurred. The first is evidence of the involvement of oxidized LDL and its apolipoprotein B in atherogenesis. Research indicates that antioxidants have a suppressive effect on atherogenesis when oxidized LDL has been involved in lesion development. The data linking the development of autoimmune reactions to these oxidatively altered lipoproteins are also impressive. Further, there is increasing evidence that atherogenesis in nonhuman primates and in people in whom chronic sustained circulating immune complexes are involved is likely to be accelerated, even when few or no classic risk factors are present. These lesions appear to represent a distinct microarchitectural form of concentric and transmural atherosclerosis that is better classified as "atheroarteritis."